Braz J Med Biol Res 35(2) 2002 Brazilian Journal of Medical and Biological Research (2002) 35: 153-159 ISSN 0100-879X Microsatellite instability and cytogenetic survey in myeloid leukemias 1 Departamento de Genética, Universidade Federal do Paraná, Curitiba, PR, Brasil 2 Departamento de Hematologia-Oncologia, Hospital Universitário de Santa Maria, Santa Maria, RS, Brasil 3 Hemocentro de Pernambuco, Recife, PE, Brasil 4 Unidade de Transplante de Medula Óssea, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, PR, Brasil E.M.S.F. Ribeiro 1 , J.M. Rodriguez 1 , V.M. Cóser 2 , M.G. Sotero 3 , J.M. Fonseca Neto 3 , R. Pasquini 4 and I.J. Cavalli 1 Abstract Microsatellites are short tandem repeat sequences dispersed through- out the genome. Their instability at multiple genetic loci may result from mismatch repair errors and it occurs in hereditary nonpolyposis colorectal cancer. This instability is also found in many sporadic cancers. In order to evaluate the importance of this process in myeloid leukemias, we studied five loci in different chromosomes of 43 patients, 22 with chronic myelocytic leukemia (CML) in the chronic phase, 7 with CML in blast crisis, and 14 with acute myeloid leukemia (AML), by comparing leukemic DNA extracted from bone marrow and constitutional DNA obtained from buccal epithelial cells. Only one of the 43 patients (2.1%), with relapsed AML, showed an altera- tion in the allele length at a single locus. Cytogenetic analysis was performed in order to improve the characterization of leukemic sub- types and to determine if specific chromosome aberrations were associated with the presence of microsatellite instability. Several chromosome aberrations were observed, most of them detected at diagnosis and during follow-up of the patients, according to current literature. These findings suggest that microsatellite instability is an infrequent genetic event in myeloid leukemias, adding support to the current view that the mechanisms of genomic instability in solid tumors differ from those observed in leukemias, where specific chro- mosome aberrations seem to play a major role. Correspondence E.M.S.F. Ribeiro Departamento de Genética, UFPR Caixa Postal 19071 81531-970 Curitiba, PR Brasil Fax: +55-41-266-2042 E-mail: [email protected]Research partially supported by CNPq. Received March 8, 2001 Accepted November 7, 2001 Key words • Genomic instability • Mismatch repair errors • Cytogenetics • Leukemogenesis Introduction Microsatellite instability (MSI) consti- tutes a recognized mechanism of mutation involved in human cancer. Differences in nucleotide repeats, representing either an expansion or a reduction of these sequences, were initially described in hereditary non- polyposis colorectal cancer (HNPCC) (1) and have been found in HNPCC-related can- cer and sporadic tumors in other organs (re- viewed in 2 and 3). MSI appears to reflect multiple replication errors because of defec- tive mismatch repair genes. Five products have been shown to be involved in human cells (MSH2, MSH6, MSH3, MLH1 and PMS2) (4) and data regarding MSI in human hematologic diseases are unclear. Wada et
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Braz J Med Biol Res 35(2) 2002
Genomic instability in leukemiasBrazilian Journal of Medical and Biological Research (2002) 35: 153-159ISSN 0100-879X
Microsatellite instability andcytogenetic survey in myeloidleukemias
1Departamento de Genética, Universidade Federal do Paraná, Curitiba, PR, Brasil2Departamento de Hematologia-Oncologia, Hospital Universitário de Santa Maria,Santa Maria, RS, Brasil3Hemocentro de Pernambuco, Recife, PE, Brasil4Unidade de Transplante de Medula Óssea, Hospital de Clínicas,Universidade Federal do Paraná, Curitiba, PR, Brasil
E.M.S.F. Ribeiro1,J.M. Rodriguez1,
V.M. Cóser2, M.G. Sotero3,J.M. Fonseca Neto3,
R. Pasquini4 andI.J. Cavalli1
Abstract
Microsatellites are short tandem repeat sequences dispersed through-out the genome. Their instability at multiple genetic loci may resultfrom mismatch repair errors and it occurs in hereditary nonpolyposiscolorectal cancer. This instability is also found in many sporadiccancers. In order to evaluate the importance of this process in myeloidleukemias, we studied five loci in different chromosomes of 43patients, 22 with chronic myelocytic leukemia (CML) in the chronicphase, 7 with CML in blast crisis, and 14 with acute myeloid leukemia(AML), by comparing leukemic DNA extracted from bone marrowand constitutional DNA obtained from buccal epithelial cells. Onlyone of the 43 patients (2.1%), with relapsed AML, showed an altera-tion in the allele length at a single locus. Cytogenetic analysis wasperformed in order to improve the characterization of leukemic sub-types and to determine if specific chromosome aberrations wereassociated with the presence of microsatellite instability. Severalchromosome aberrations were observed, most of them detected atdiagnosis and during follow-up of the patients, according to currentliterature. These findings suggest that microsatellite instability is aninfrequent genetic event in myeloid leukemias, adding support to thecurrent view that the mechanisms of genomic instability in solidtumors differ from those observed in leukemias, where specific chro-mosome aberrations seem to play a major role.
Microsatellite instability (MSI) consti-tutes a recognized mechanism of mutationinvolved in human cancer. Differences innucleotide repeats, representing either anexpansion or a reduction of these sequences,were initially described in hereditary non-polyposis colorectal cancer (HNPCC) (1)
and have been found in HNPCC-related can-cer and sporadic tumors in other organs (re-viewed in 2 and 3). MSI appears to reflectmultiple replication errors because of defec-tive mismatch repair genes. Five productshave been shown to be involved in humancells (MSH2, MSH6, MSH3, MLH1 andPMS2) (4) and data regarding MSI in humanhematologic diseases are unclear. Wada et
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al. (5) reported a high frequency (53%) ofMSI in the evolution of chronic myelocyticleukemia (CML) from chronic phase to blastcrisis and concluded that this loss of fidelityin replication and repair mechanism may beassociated with CML evolution. However,other studies have shown that instability isinfrequent in the evolution of CML to blastcrisis (6,7) and in acute leukemias (8-12).On the other hand, cytogenetic analysis is ofrecognized importance in leukemias sincethe years before the availability of bandingtechniques, but few studies have discussedboth mechanisms together. We report here astudy of MSI and cytogenetics in 43 patientswith different types of myeloid leukemias.
Material and Methods
Clinical samples
Bone marrow samples were obtained from36 untreated patients, 22 with CML in thechronic phase and 14 with acute myeloidleukemia (AML), and from 7 patients withCML in blast crisis previously treated for thechronic phase. Thirty samples were collectedat the Department of Hematology, Univer-sity Hospital, Federal University of Paraná(UFPR), Curitiba, PR, 10 were collected atthe Department of Hematology-Oncology,University Hospital, Santa Maria, RS, bothin South of Brazil, and 3 were collected atthe Blood Center of Recife, PE, NortheastBrazil. The samples were collected in 1996and 1997. During this period, all patientswhose bone marrow samples were sent tothe Cytogenetics Unit (UFPR) were consid-ered. On the occasion of the next ambulatoryvisit of the patient, following approval by thereferring physician, informed consent wasobtained and buccal epithelial cells werecollected as a source of constitutional DNA.For the patients admitted to the hospital,consent was obtained on the ward. The studywas approved by the Hospital Ethics Com-mittee (UFPR).
Samples for which it was not possible toobtain amplification products of a minimumof two loci were excluded from the study(two from patients with blast crisis and onefrom a patient with AML). We believe that,within the defined period of time for samplecollection from patients referred to the He-matology Department, the data were fullyrandomized, without any bias. At the othertwo centers where samples were obtainedthe same selection criteria were used. Fif-teen cases were received from the BloodCenter of Pernambuco; however, only threecases were feasible for DNA extraction.
The types of leukemias were diagnosedaccording to the French-American-British(FAB) classification (13) using immunohis-tochemistry, immunophenotyping and cyto-genetics.
Cytogenetic analysis
Bone marrow aspirates were processedby the method of Williams et al. (14), Gbanding was performed by the method ofScheres (15), and karyotypes were describedaccording to the International System forHuman Cytogenetic Nomenclature (16).
DNA isolation
Genomic DNA from bone marrow sam-ples and buccal epithelial cells was obtainedby phenol-chloroform extraction followingproteinase K treatment, as previously de-scribed (17).
Microsatellite amplification
Polymerase chain reactions (PCR) wereperformed on 50 µl of reaction mixture con-taining 50 mM KCl, 2.0 mM MgCl2, 10 mMTris, pH 8.3, 200 µM of each of the fourdNTPs, 0.25 µM of each primer, 12.5-50 ngof genomic DNA, and 2.5 U of Taq poly-merase (Gibco-BRL, Gaithersburg, MD,USA). Samples were processed through 28
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cycles at 94ºC for 30 s, 62ºC for 30 s and72ºC for 2 min in a Perkin Elmer/Cetusthermal cycler 9600 (Norwalk, CT, USA).
Single-strand conformation polymorphism
Amplified PCR fragments were diluted1:1 in denaturing loading buffer consistingof 98% formamide, 10 mM EDTA, pH 8.0,0.02% bromophenol blue, and 0.02% xylenecyanol FF, and denatured at 94ºC for 5 min.The denatured and non-denatured reactionswere subjected to electrophoresis side byside in a vertical discontinuous polyacryl-amide gel system (18). A non-denaturedsample was used to control for the presenceof technical artifacts, which could lead to thefalse conclusion of the presence of new alle-les. Electrophoresis was run overnight at 10W (room temperature) and bands were de-tected by silver staining (19).
Analysis of microsatellite instability
We examined the extracted DNA for ge-netic alterations at five separate loci contain-ing nucleotide repeat sequences. Informa-tion on repeat sequences, chromosome lo-calization, length of amplified fragments andprimer sequences, obtained from the Ge-nome Database (20), are indicated in Table1. MSI was analyzed by comparison of DNA-banding patterns of PCR products of micro-
satellite sequences in leukemic and matchednormal buccal epithelial cells.
Statistical analysis
The detection of MSI as a function of thenumber of analyzed loci was evaluated bythe regression coefficient.
Results and Discussion
Alterations in microsatellite markers havebeen implicated as a frequent feature inHNPCC (75-78%) (1) and in some sporadiccancer (3-30%) (2,3). The data for leuke-mias are contradictory, ranging from zero to94% in different reports (5-12,21-32). Herewe report a study on 43 patients with CMLand AML. Patients’ characteristics are de-scribed in Table 2. Of 207 paired PCR reac-tions, 178 were informative (86%). In theother 29 samples, amplification failed orDNA was not sufficient. Only one patientwith relapsed AML showed instability at asingle locus (2.3%) as indicated in Table 2and Figure 1.
Most reports did not show instability dur-ing the chronic phase of CML (5-7,9,21) andour results agree with them. Although theinstability of microsatellites is implicated inthe evolution from chronic phase to blastcrisis (5), other authors (6,7) have not con-firmed these results, and the present report is
Table 1. Synthetic oligonucleotides used in this study.
Marker Chromosomal Amplicon size Core repeat Nucleotide sequencelocation range (bp) unit of primers (5'-3')
+der(22)t(9;22)28 M/21 CML-BC (-) (-) (-) (-) (-) ND Ct29 M/32 CML-BC (-) (-) (-) (-) F +8,t(9;22)(q34;q11),+19, Death
+der(22)t(9;22)30 M/57 AML-M5b-Relapse (-) (-) (-) (+) F ND Death31 F/9.10 AML-M3 (-) (-) (-) (-) (-) t(15;17)(q22;q11) Death32 F/11 ALL L1 - pre-B (-) (-) (-) (-) (-) t(1;19)(q23;p13), del(15)(q22) Relapse - Ct33 M/21 ALL L1 - B (-) (-) (-) (-) F +21 Death34 M/14 AML-M2 (-) (-) (-) (-) (-) +8 Relapse - Ct35 M/35 ALL pre-B (-) (-) (-) (-) (-) 46,XY Death36 M/16 ALL L1 - T (-) (-) (-) (-) (-) ND Death37 F/20 AML-M3 (-) (-) F (-) F ND Death38 F/35 AML-M3 (-) (-) F (-) F t(15;17)(q22;q11) Autologous BMT39 F/39 AML-M4 (-) (-) F (-) (-) t(17;19)(q22;p13) Death40 M/41 AML post-MDS (-) (-) (-) (-) (-) -7,+mar Death41 M/28 AML-M0 (-) (-) (-) (-) (-) del(5)(p13) Autologous BMT42 M/8.6 AML-M2 (-) (-) (-) (-) (-) t(9;11)(q22;q23) Death43 M/16 ALL L1 - pre-B (-) (-) (-) (-) F +mar Allogeneic BMT44 F/53 AML-M4 (-) (-) (-) (-) (-) 46,XX Relapse - death45 F/59 AML (-) (-) (-) (-) (-) 46,XX Death46 F/15 AML-M2 F (-) (-) F F +5,+8,t(8;21)(q22;q22),+9,-17, Death
+der(21)t(8;21)47 F/72 AML post-MDS (-) (-) F F ND ND Death48 F/35 AML-M4 (-) (-) (-) (-) (-) del(7)(q32),+mar Allogeneic BMT
(-) = no microsatellite instability, (+) = microsatellite instability, F = failure in amplification, ND = not done, CML = chronic myeloid leukemia, CP =chronic phase, BC = blast crisis, AML = acute myeloid leukemia, ALL = acute lymphocytic leukemia, Ct = chemotherapy, BMT = bone marrowtransplantation.
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in the same direction, since no alterations inelectrophoresis pattern were observed among7 patients. With respect to the patients withacute leukemias, we studied 14 patients withAML (10 de novo, 1 relapse, 1 secondary toprevious acute lymphoblastic leukemia and2 post-myelodysplastic syndrome). The onlycase with alteration in the allele length at asingle locus (SE33) was the patient in re-lapse (patient number 30) (7.14%). Tasakaet al. (10) studied the progression of AMLand found 12% MSI in the diagnostic andremission phase in contrast to 35% in therelapsed phase, indicating that mismatch re-pair errors might be of importance during theprogression of the disease. Ben-Yehuda etal. (23) found the highest index described(94%) in patients with therapy-related AML,suggesting a mutator phenotype. Anotherstudy, however, concluded that MSI is infre-quent in myeloid leukemias. Boyer et al. (11)investigated MSI in the transformation ofmyeloid cells to myelodysplastic syndromeand/or AML using a panel of 14 microsatel-lite loci. Only one example of MSI wasfound in 48 patients. Rimsza et al. (12) retro-spectively studied 132 AML cases in adultpatients and no single case of MSI was dem-onstrated either in diagnosted or relapsedcases. Sill et al. (24) studied 20 patientsusing a panel of 12 microsatellite loci anddetected no alterations.
In view of that we analyzed a small num-ber of markers, we checked if this factorcould be responsible for the absence of in-stability in our study. Using the informationfrom 19 papers (Table 3) and our results, theregression coefficient showed a b value =-0.0011, t = -0.786, and P>0.40, showingthat the frequencies observed are independ-ent of the number of loci analyzed. This is inagreement with several investigators, whothink that for an MSI+ pattern to be defined,alterations in just one microsatellite markermay be sufficient. Despite the presence ofthousands of microsatellite loci throughoutthe genome, there is no indication from any
Table 3. Frequencies of microsatellite instabilityobserved in 20 different studies.
N X Y References
1 5 0.152 Wada et al. (5)2 12 0.000 Silly et al. (6)3 10 0.000 Mori et al. (7)4 11 0.029 Robledo et al. (8)5 22 0.007 Pabst et al. (9)6 69 0.004 Tasaka et al. (10)7 14 0.002 Boyer et al. (11)8 3 0.000 Rimsza et al. (12)9 1 0.043 Indraccolo et al. (21)
10 8 0.523 Ben-Yehuda et al. (23)11 12 0.000 Sill et al. (24)12 56 0.005 Baccichet et al. (25)13 20 0.017 Fennelly et al. (26)14 85 0.001 Takeuchi et al. (27)15 5 0.010 Reato et al. (28)16 6 0.000 Hayami et al. (29)17 16 0.016 Tasaka et al. (30)18 11 0.250 Fu et al. (31)19 5 0.032 Tanosaki et al. (32)20 5 0.005 Present study
N = number of the study; X = number of locistudied; Y = frequency of instability observed/number of reactions performed.
BM NFigure 1. Alterations in the elec-trophoretic pattern observed inpatient number 30, locus SE33.Arrows show shift or extrabands. BM = bone marrow; N =normal.
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published report to date that studies based onscreening of three microsatellite markers areany less accurate than those using four ormore markers (3). Another fact to be consid-ered is the type of marker. Boland et al. (33)defined a panel of five microsatellite mark-ers recommended as a reference for futureMSI studies in colorectal cancer. Unfortu-nately, there is no similar recommendationfor leukemias and more data will be neces-sary to define a basic panel.
With respect to cytogenetic analysis, allthe 22 patients with CML in the chronicphase showed the t(9;22) mutation (Ph+)and only one had additional chromosomeaberrations (number 22). This patient wasclinically and hematologically in the chronicphase, but cytogenetic analysis showed tri-somy of chromosome 8 and he progressed toblast crisis and died within a short period oftime. As expected, additional cytogeneticaberrations were frequently observed in thegroup of patients with blast crisis. Amongthe 14 patients with AML several chromo-some aberrations were detected, some ofthem related to the FAB subgroup (Table 2).
Independent of microsatellite analysis,we could establish relations between chro-
mosome aberrations and follow-up for mostof the patients. For example, in AML, tri-somy of chromosome 8 (patient number 32)has been reported to be associated with a lownumber of complete remissions. The t(15;17)(q22;q12) translocation is related to a highnumber of remissions with the combinationof ATRA and chemotherapy, but the pa-tients are at high risk for early death fromintracranial hemorrhage, which was the causeof death of patient number 31. Monosomy 7(patient number 36) and t(9;11)(p21-22;q23)(patient number 38) have been reported to beassociated with a poor prognosis. These cor-relations have been extensively described inthe literature and are used to guide the choiceof the best treatment. Based on these consid-erations, we conclude that cytogenetics isalready the method of choice for prognosisand for treatment indications at the time ofdiagnosis. For follow-up, molecular cytoge-netics and the detection of genic rearrange-ments by PCR are recognized as better meth-ods than traditional cytogenetics to evaluateminimal residual disease. In contrast, micro-satellite analysis does not seem to contributeto the diagnosis or follow-up of leukemicpatients.
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